1. Field of the Invention
The present invention relates to a protease which efficiently cleaves the pro-structure part of pro-transglutaminase resulting in an active transglutaminase, and to a nucleic acid encoding the protease, wherein the pro-transglutaminase is produced by actinomycetes. The present invention also relates to a method of producing microbial transglutaminase in its active form using the protease. Additionally, the present invention relates to a method of producing a neutral metalloprotease.
2. Brief Description of the Related Art
Transglutaminase is an enzyme which catalyzes the acyltransfer reaction of γ-carboxylamide groups in the peptide chain of the protein. When the enzyme reacts with a protein, the formation of the cross-linkage ε-(γ-Glu)-Lys, and the replacement of Gln with Glu by deamidation can occur. Transglutaminases have been used to manufacture gelled food products such as jelly, yogurt, cheese, or gelled cosmetics and others, and to improve the quality of meat, etc. (Japanese publication of examined application (JP-Kokoku) No. 1-50382). Moreover, transglutaminase is highly useful in industry in that it has been used to manufacture materials for thermostable microcapsules, carriers for immobilized enzyme, etc.
Expression of animal transglutaminase activity is calcium-dependent, and transglutaminases from microorganisms (microbial transglutaminase(s), which is/are also referred to as “MTG(s)” hereinafter), have been previously reported to also be calcium-independent. A microbial transglutaminase from a bacterium belonging to genus Streptoverticillium has been reported. Such Streptoverticillium bacteria include, for example, Streptoverticillium griseocarneum IFO 12776, Streptoverticillium cinnamoneum sub sp. cinnamoneum IFO 12852, Streptoverticillium mobaraense (hereinafter, S. mobaraense) IFO 13819, and others (Publication of unexamined Japanese patent application (JP-Kokai) No. 64-27471).
Because these transglutaminases, however, have been produced via purification from cultures of the microorganisms described above, problems have been reported regarding the produced amount, production efficiency, and the like. Then, in an attempt to more efficiently secrete heterologous proteins, a method was reported using a coryneform bacterium as a host, and a fused protein whereby transglutaminase was connected downstream of the signal peptide domain of the coryneform bacterium, and the transglutaminase was efficiently secreted resulting in a high yield of transglutaminase (WO 01/23591). In this study, a method is also described wherein inactive MTG is secreted as pro-transglutaminase (referred to as “pro-MTG” hereinafter) whereby a pro-structure part is connected to MTG, and then this pro-structure part is cleaved by a protease to convert it into an active transglutaminase. A further method is described wherein an active transglutaminase is directly produced in a culture medium by co-expressing SAM-P45, which is a serine protease derived from actinomycetes, in a sufficient amount in a coryneform bacterium which also produces the pro-MTG.
Although a method in which an active transglutaminase is directly produced by co-expressing pro-MTG and a protease which allows cleavage of the pro-structure part of the pro-MTG in a coryneform bacterium is assumed to be an extremely efficient method of producing transglutaminase, the substrate specificity of SAM-P45 is not very strict, and it may digest and degrade not only the pro-structure part of the pro-MTG but also the transglutaminase itself to some degree. Therefore, the handling of SAM-P45 may not be easy. When SAM-P45 is used, therefore, the production method of transglutaminase should be strictly controlled such that degradation of the transglutaminase in the culture medium will not occur.
There is still, therefore, demand for a protease which can selectively cleave only the pro-structure part of pro-MTG, with as little degradation of the transglutaminase itself as possible during the production of an active transglutaminase.
A dispase derived from Bacillus polymyxa is known (Eur. J. Biochem., vol. 257, p. 570-576 (1998)) to be an enzyme besides SAM-P45 which cleaves the pro-structure part of pro-MTG. A large amount of the enzyme, however, is required to cleave the pro-structure part, and there is a risk of degrading the transglutaminase itself. In addition, dispase is a reagent in cell culture, so it is expensive when used an enzyme for industrial use.